This project investigates the regulation of metabolic flux in the hepatic tricarboxylic acid (TCA cycle) and its role in hepatic insulin resistance. The proposal follows up on key findings in the last funding cycle which indicate that anaplerotic/cataplerotic biosynthetic flux from the TCA cycle and oxidative flux of the TCA cycle are reciprocally regulated and that these pathways are elevated during hepatic insulin resistance. These findings challenge the role of impaired oxidative metabolism as an instigator of hepatic insulin resistance. Thus, we propose a novel hypothesis that elevated hepatic TCA cycle flux is a principal metabolic mediator of pathologies of hepatic insulin resistance by potentiating biosynthetic flux (e.g. gluconeogenesis) and oxidative stress. We test this hypothesis using conditional KO mice, state of the art 13C and 2H tracer based NMR and MS/MS isotopomer methods to evaluate hepatic metabolic fluxes of glucose, lipid and TCA cycle metabolism, and standard evaluation of signaling, gene expression and insulin sensitivity. The first aim is to test whether insulin action mediates dys/regulation of TCA cycle fluxes. We determine if acute genetic excision of insulin signaling recapitulates or exasperates elevated TCA cycle flux in HFD mice and whether acute deletion of FOXo1 ameliorates TCA cycle flux and oxidative stress during a HFD. Since these pathways are also subject indirect upregulation by substrate delivery during insulin resistance, the second aim is to determine the mechanism of co-regulation between cataplerotic biosynthesis and oxidative TCA cycle flux. While the acute regulation of these pathways by ATP, NADH and allosteric regulators are known, we will test whether AMPK and Sirt3 act as acute molecular regulators of flux through TCA cycle pathways. Finally, if elevated TCA cycle flux potentiates metabolic pathologies of hepatic insulin resistance, then suppressing TCA cycle pathways should prevent the onset of hepatic complications. Thus, the third aim is to determine if genetic suppression of the cataplerotic or the oxidative span of the TCA cycle results in improved metabolic, oxidative stress and inflammatory response to insulin resistance.